1. Field of the Invention
The present invention relates to blood assays, and, more particularly, to methods for assaying thrombin generation, and, most particularly, to methods for assaying thrombin generation in a whole blood sample.
2. Description of Related Art
The coagulation of blood occurs through a complex series of reactions that function as a biological amplifier and culminate in the conversion of soluble circulating fibrinogen into a fibrin meshwork at the site of a vascular injury, providing stability to a hemostatic plug of platelets. In this system, relatively few initiating substances sequentially and proteolytically activate a cascade of circulating precursor proteins, the coagulation factor enzymes.
Among the reactions is the conversion of prothrombin to thrombin, which is the pivotal enzyme of the coagulation system. Thrombin is a serine protease that rapidly activates platelets and converts fibrinogen to insoluble fibrin. Thrombin also converts FXIII to FXIIIa, which chemically cross-links the fibrin clot.
Abnormalities in the coagulation cascade can have potentially fatal effects, leading to extremes of bleeding disorders and excessive clotting, thrombosis.
It is known to assess the coagulation system by activating the cascade and measuring the time it takes for the sample blood or plasma to clot. Although clotting times provide clinically useful information, they actually only represent the initial (<5%) thrombin generation. The majority of thrombin is formed after this initial period.
Attempts have been made to quantify the dynamics of thrombin formation. Hemker et al. have devised a commonly used technique (“Thrombin Generation in Plasma: Its Assessment via the Endogenous Thrombin Potential,” Thrombosis and Haemostasis 74, 134-38, 1995; “Continuous Registration of Thrombin Generation in Plasma, Its Use for the Determination of the Thrombin Potential, Thrombosis and Haemostatis 70, 617-24, 1993; “The Thrombogram: Monitoring Thrombin Generation in Platelet Rich Plasma,” Thrombosis and Haemostasis 83, 589-91, 2000; “Calibrated Automated Thrombin Generation Measurement in Clotting Plasma,” Pathophysiology Haemostasis and Thrombosis 33, 4-15, 2003; the contents of all of which are incorporated hereinto by reference).
The techniques of Hemker et al. include the addition of a thrombin activator to a plasma sample together with a fluorogenic thrombin substrate. Thrombin formed during the clotting reaction consumes the substrate, producing a conversion product that is detected fluorometrically in real time. From these data can be calculated the endogenous thrombin potential (ETP), which indicates how much thrombin has been active and for how long. The data can also be used to calculate a lag time (the time to formation of thrombin), the maximal thrombin concentration reached, and the time to the peak thrombin formation.
What has not been successfully achieved, however, is the measurement of thrombin generation in whole blood, primarily owing to fluorescence signal quenching by red blood cells.